90 YEARS OF PROGESTERONE Progesterone receptor signaling in the normal breast and its implications for cancer

Progesterone is considered as the pregnancy hormone and acts on many different target tissues. Progesterone receptor (PR) signaling is important for normal development and the physiologic function of the breast and impinges on breast carcinogenesis. Both systemically and locally, in the breast epithelium, there are multiple layers of complexity to progesterone action, many of which have been revealed through experiments in mice. The hormone acts via its receptor expressed in a subset of cells, the sensor cells, in the breast epithelium with different signaling outcomes in individual cells eliciting distinct cell-intrinsic and paracrine signaling involving different mediators for different intercellular interactions. PR expression itself is developmentally regulated and the biological outcome of PR signaling depends on the developmental stage of the mammary gland and the endocrine context. During both puberty and adulthood PR activates stem and progenitor cells through Wnt4-driven activation of the myoepithelium with downstream Adamts18-induced changes in extracellualr matrix (ECM) / basal membrane (BM). During estrous cycling and pregnancy, the hormone drives a major cell expansion through Rankl. At all stages, PR signaling is closely tied to estrogen receptor a (ER) signaling. As the PR itself is a target gene of ER, the complex interactions are experimentally difficult to dissect and still poorly understood. Ex vivo models of the human breast and studies on biopsy samples show that major signaling axes are conserved across species. New intraductal xenograft models hold promise to provide a better understanding of PR signaling in the normal breast epithelium and in breast cancer development in the near future.

Role of canonical Wnt signaling in mammary gland development

Renuga Devi Rajaram

The female reproductive hormones estrogens, progesterone, and prolactin control postnatal breast development and are important to breast carcinogenesis. In particular, exposure to progesterone is tightly linked to breast cancer risk. Using the mouse as a model system to study the mechanisms through which hormones elicit morphogenetic changes in the mammary gland in vivo, we demonstrated that progesterone acts by a paracrine mechanism and identified Wnt4 as a target of progesterone important in mediating side branch formation. The mechanisms underlying Wnt4 function remains poorly understood. To understand the pattern of canonical Wnt signaling activation during mammary gland development and to address which cellular compartment of the mammary gland is activated by canonical Wnt signaling, a reporter mouse model was utilized in which the lacZ gene is under the control of endogenous Axin2 promoter, classical target promoter of canonical Wnt signaling. Analysis of reporter mice at different developmental stages of mammary gland development showed the highest reporter activity during early pregnancy which correlated with the time of side branch formation. At this stage, β-galactosidase activity was found exclusively in myoepithelial cells. Overexpression of Wnt1 from MMTV promoter increases reporter activity in the myoepithelium. Gene expression analysis on FACS sorted luminal and myoepithelial cells shows that known canonical Wnt signaling pathway components such as receptors and co receptors as well as targets are specifically expressed in the myoepithelial compartment further supporting the notion that myoepithelial cells are the target cells for Wnt1 and Wnt4 secreted by luminal cells. Consistent with a model whereby progesterone induced Wnt4 secretion results in canonical Wnt signaling activation, reporter activity was induced by progesterone treatment and lacZ expression increased during diestrus when serum progesterone levels are high under physiological conditions. Analysis of contralateral mammary glands engrafted with PR-/-.Axin2 lacZ and PR+/-.Axin2 lacZ epithelium during pregnancy revealed that intact PR signaling is required to activate canonical Wnt signaling. Similar experiments with Wnt4-/-.Axin2 lacZ and Wnt4+/+.Axin2 lacZ mammary epithelium showed that reporter activation relies on Wnt4. Histological analysis on mammary glands in which β-catenin was ablated using MMTV-Cre revealed that β-catenin deleted cells were present only in the luminal epithelial compartment and showed that β-catenin deleted myoepithelial cells cannot participate in side branch formation. Similarly, ectopic expression of dominant negative β-catenin in wt MECs reduced side branching indicating that canonical Wnt signaling mediated by β-catenin is required for side branching. Furthermore, constitutive activation of β-catenin in the mammary epithelium was sufficient to induce side branch formation in virgin mice. In Wnt4 mutant epithelium, proliferation of progesterone receptor positive and negative cells were significantly reduced in response to progesterone treatment. Wnt signaling has been implicated in stem/progenitor activation in several tissues including mammary gland. Consistent with Wnt4 activating stem/progenitors, serial transplantation assays revealed that Wnt4-/- epithelium failed to reconstitute already at 2nd to 3rd generation when the wt has the ability to reconstitute up to seven generations indicating that Wnt4 is required for stem cell function while Wnt4 is dispensable for embryonic and rudimentary mammary gland formation. Histology and gene expression analysis in late pregnancy showed that Wnt4 mutant epithelia were able to differentiate and produce milk proteins, further supporting the thought that Wnt4 function is important for stem/progenitor cell activity and not required for differentiation. Taken together, I report that Wnt4 induces canonical Wnt signaling in myoepithelial cells and activation of canonical Wnt signaling is required and sufficient for side branching. Moreover, Wnt4 function is essential for mammary stem/progenitor cells.

EPFL2010

Stem cells and the stem cell niche in the breast: an integrated hormonal and developmental perspective

Cathrin Brisken

The mammary gland is a unique organ in that it undergoes most of its development after birth under the control of systemic hormones. Whereas in most other organs stem cells divide in response to local stimuli, to replace lost cells, in the mammary gland large numbers of cells need to be generated at specific times during puberty, estrous cycles and pregnancy to generate new tissue structures. This puts special demands on the mammary stem cells and requires coordination of local events with systemic needs. Our aim is to understand how the female reproductive hormones control mammary gland development and influence tumorigenesis. We have shown that steroid hormones act in a paracrine fashion in the mammary gland delegating different functions to locally produced factors. These in turn, affect cell-cell interactions that result in changes of cell behavior required for morphogenesis and differentiation. Here, we discuss how these hormonally regulated paracrine interactions may impinge on stem cells and the stem cell niche and how this integration of signals adds extra levels of complexity to current mammary stem cell models. We propose a model whereby the stem cell niches change depending on the developmental stages and the hormonal milieu. According to this model, repeated hormone stimulation of stem cells and their niches in the course of menstrual cycles may be an important early event in breast carcinogenesis and may explain the conundrum why breast cancer risk increases with the number of menstrual cycles experienced prior to a first pregnancy.

Humana Press Inc, Springer2007

Mechanisms underlying cell-fate determination in the mammary gland development

Duje Buric

Notch signaling pathway is an important developmental pathway and has been implicated in both mammary gland development and tumorigenesis. Several studies investigating Notch signaling in mouse mammary gland implied that Notch signaling is an important factor in the determination of the luminal cell type. Clinical studies on breast tumors as well as studies in transgenic mouse models overexpressing active Notch receptors suggested an oncogenic function of Notch in the mammary gland connecting it to the poor breast cancer prognosis. However the physiological role of Notch signaling and its downstream mechanisms remain unclear. Analysis of Transgenic-EGFP Notch reporter mouse mammary epithelium revealed that Notch signaling is active specifically in a subset of hormone receptor positive cells. More sensitive FACS analysis consistently shows EGFP expression in HR+ luminal cells and reveals a weaker signal in a subset of basal (CD24lo) cells. Additionally mRNA analysis revealed that Notch active luminal cells are expressing Wnt4 ligand. To unveil the role of the Notch signaling in the Wnt4 expressing HR+ cells, we conditionally deleted RBP-Jκ, Notch signaling mediator, in Wnt4 expressing subpopulation of hormone receptor positive cells via Wnt4Cre. Abrogation of RBP-Jκ resulted in loss of progesterone receptor expression in 90% of cells bearing the RBP-Jκ deletion while estrogen receptor expression remained intact, implicating RBP-Jκ in regulation of progesterone receptor expression. To test whether Notch signaling regulates PR expression in the entire HR+ population, we analyzed mammary epithelium in which RBP-Jκ has been conditionally deleted via MMTV-Cre in progenitor cells revealing the presence of a subpopulation of luminal hormone receptor positive cells that differentiate independently of Notch signaling. Time directed deletion of RBP-Jκ via intraductally injected Adeno-Cre virus into the adult mouse mammary ductal system through the nipple additionally confirmed that RBP-Jκ is required for progesterone receptor expression. Chromatin immunoprecipitation assay showed that RBP-Jκ binds two out of four putative RBPJκ binding sites in the progesterone receptor promoter. Inhibition of Notch signaling in vivo via intraductal injection of γ-secretase inhibitor, DAPT, resulted in reduced progesterone receptor expression strongly suggesting that Notch-related RBP-Jκ signaling is responsible for PR expression. In this project we are proposing that there are two different populations of HR+ cells: one defined as Wnt4 expressing subpopulation of HR+ cells in which PR expression is dependent of Notch signaling, and another one defined as Wnt4 non expressing subpopulation of HR+ cells which is independent of Notch signaling. Since Notch signaling is able to replace ER signaling and activate ER target genes in the endocrine therapy resistant cell lines, Wnt4 expressing population of HR+ luminal cells might play a crucial role in the acquiring of the resistance.